Cell Tissue Kinet. (1975) 8, 51-60.

RECOVERY OF PROLIFERATING HAEMOPOIETIC PROGENITOR CELLS AFTER KILLING BY HYDROXYUREA G . S . HODGSON,T. R. BRADLEY,*R. F. MARTIN,M. SUMNER* A N D P. FRY* Biological Research Unit, Cancer Institute, Melbourne, and *Physiology Department, University of Melbourne (Received 14 May 1974; revision received 27 June 1974)

ABSTRACT

Two doses of 1 mg/g of hydroxyurea (HU), injected 7 hr apart into irradiated mice in which CFU-S were proliferating during marrow regeneration, killed about 90 % of CFU-S. This same dose regime injected into normal female mice, with nonproliferating CFU-S killed 92 % of CFU-C, 99 of ESC and only 30 % of CFU-S. One day afterthe treatment CFU-S had decreased to 50 "/,and remained at about this level for a further day then returned to normal values. In spleen the increase in CFU-S was delayed by a day and showed a marked overshoot. During the period that CFU-S were decreased in number they were actively proliferating. Marrow CFU-C recovered in an exponential manner with a doubling time of 16 hr. Spleen CFU-C recovered 1 day later than marrow and showed a pronounced overshoot. ESC recovered very rapidly with doubling time of 5 hr. The changes in 59Fe incorporation into RBC, and the peripheral blood picture, were a delayed reflection of the changes in ESC and CFU-C. INTRODUCTION The myelopoietic component of the haemopoietic system is postulated (McCulloch & Till, 1972) to contain the following categories of cells: (a) Pluripotential stem cells capable of extensive self renewal and of differentiation into erythropoietic, granulocytic and megakaryocytic cells. The stem cells are operationally defined by their capacity to give rise to discrete colonies in the spleen when injected into lethally irradiated mice (Till & McCulloch, 1961) and are referred to as CFU-S. In the normal adult mouse only about 10 % of CFU-S are in DNA-S phase of cell cycle at any given time. (b) Morphologically unrecognizable progenitors of the mature blood cells, which are regarded as progeny of pluripotential stem cells but committed to a differentiation course. Two of the committed progenitors can be operationally defined: (i) Progenitors of granulocytes and macrophages, which give rise to colonies of granulocytes and macrophages in culture (Bradley & Metcalf, 1966), referred to here as CFU-C. (i) Progenitors of erythrocytes which can give rise to erythroid colonies in culture (Stephenson et al., 1971) and to a wave of Correspondence: Dr G. S. Hodgon, Biological Research Unit, Cancer Institute, 481 Little Londsdale St., Melbourne 3000, Australia. 51

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G . S. Hodgson et al.

erythropoiesis in the spleen of a RBC transfused mouse after injection of erythropoietin (Filmanowicz & Gurney, 1961). These progenitors are referred to as erythropoietin sensitive cells (ESC). Both CFU-C (Iscove, Till & McCulloch, 1970) and ESC (Hodgson, 1967) are actively proliferating in normal mice. On the basis of the difference in cycle properties of CFU-S with those of CFU-C and ESC it should be possible to devise a dose schedule of a cycle specific drug, which kills only proliferating cells so that most of the CFU-C and ESC are killed, and the majority of the nonproliferating CFU-S survive. Hydroxyurea (HU) is a drug which preferentially kills cells in S phase (Sinclair, 1967; Phillips et ul., 1967), and has the added advantage of acting as a synchronizing agent by blocking the passage of G, cells into S phase (Sinclair, 1967). This paper describes the time course of changes in CFU-S, CFU-C and ESC in bone marrow and spleen that occur after two injections of HU given 7 hr apart. Such a dose schedule kills over 90 % of CFU-C and ESC, initially leaving 70 % of CFU-S alive. MATERIALS A N D METHODS Plasma H U concentrations were measured by the method of Nery (1966) after intraperitoneal injection of HU (Nutritional Biochemicals) 1 mg/g body weight into twenty-five 3-month-old female Balb/c mice. Groups of five mice were killed at hourly intervals. Incorporation of lZ5I-UdR(Radiochemical Centre, Amersham) into DNA was measured by the method of Martin & Hodgson (1973). DNA was estimated by the method of Martin, Donohue & Finch (1972). CFU-S assays were carried out essentially as described by Till & McCulloch (1961) using groups of at least ten 3-month-old male Balb/c mice, which had received 700 R X-irradiation (Maximar machine; 230 kV; 15 mA; dose rate 150 R/min; TSD 50; HVL 0.5 mmCu) (Hodgson, 1973). The number of bone marrow or spleen cells administered was adjusted, on the basis of preliminary experiments, so as to give approximately ten colonies per irradiated mouse spleen. CFU-C in marrow and spleen samples were assayed as described by Bradley & Sumner (1968) except that an extract of mouse placenta, membranes, pregnant uterus and embryo (PMUE) was used in sufficient quantity to evolve the maximum number of colonies as the colony stimulating factor (Bradley, Stanley & Sumner, 1971). The effectof two doses of H U on iron incorporation into RBC was determined by injection of 0-2pCi 59Fecitrate (Radiochemical Centre, Amersham) into groups of ten mice at different intervals after H U treatment and measurement of the 59Fecontent of washed RBC from 0-2 ml of blood in a Packard autogamma counter 18 hr and 48 hr after tracer injection. The effect of H U treatment on responsiveness to erythropoietin was assayed in groups of Seven female Balb/c mice 1 day after intravenous transfusion with I ml of 60 % v/v of saline washed red cells. Erythropoietin (EPO) (Step 3 lot 3001-33, Connaught Laboratories, Mellowdale, Ontario) was injected subcutaneously (3 units per mouse) at different times after H U treatment, and 48 hr later 0.2 pCi 59Fecitrate was injected intravenously; the mice were bled 24 hr after tracer injection, packed cell volume was determined and the 59Fecontent of saline washed red cells from 0.2 ml of blood measured. The PCV of the mice at the time of sampling was greater than 60 %. Transfusion was carried out only a day before the injection in order to change the ESC population as little as possible (Gregory, McCulloch &Till, 1973). With this timing the iron incorporation in transfused controls was suitably low: 0.42 % 3 days after transfusion.

Recovery of proliferating haemopoietic progenitor cells

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Reticulocytes were strained with new Methylene Blue and the fraction determined from a count of 1000 RBC. WBC were counted in a haemocytometer and the percentage of granulocytes determined by the count of 100 cells on a Giesma stained smear. RESULTS 1. Disappearance of hydroxyurea ( H U )from plasma Disappearance of H U from plasma after intraperitoneal injection of 1 mg/g HU into female Balb/c mice was exponential over two log cycles, the half disappearance was estimated by regression of log(HU) f ( t ) (r = 0.967) as 0.80 k 0.09 hr.

2. Eflect of HU on DNA synthesis, cell survival and 59Feincorporation Fig. 1 shows the dose-response curves for the effects of HU on: (1) DNA synthesis, as estimated by the incorporation of 1251-UdRinto spleen DNA of normal mice measured 1 hr after HU injection. (2) Survival of CFU-S proliferating in the spleens of lethally irradiated mice which had received 2 x lo6 isologous bone marrow cells. The first dose of HU (1 mg/g) was injected 7 days after the injection of bone marrow cells and a second (variable dose) 7 hr after the first. The spleens were removed for assay 2 hr after the second H U injection. (3) 59Feincorporation into erythrocytes of normal mice treated with a single injection of HU (1 mg/g). The tracer was injected 1 day after the injection of HU and the 59Fecontent of RBC measured 24 hr later. The doses of HU required to inhibit DNA synthesis were lower than those required either to kill cycling CFU-S or depress erythropoiesis, the latter two parameters are depressed to a similar extent by HU. In all subsequent experiments an intraperitoneal dose of 1 mg/g body weight of H U was used.

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FIG.1 . Hydroxyurea dose response. Ordinate: response as a percentage of the value obtained in uninjected controls. Abscissa : dose mg/g body weight, intraperitoneally. W, 'WUdR incorporation into DNA of mouse spleen, tracer injected 1 hr after a single injection of HU. Spleens sampled 1 hr after tracer injection. A , Survival of CFU-S(mean k SE), proliferating in the spleen of lethally irradiated mice (see Methods). e,5gFe24 hr incorporation into mouse RBC (mean It: SE) tracer injected 24 hr after HU.

G . S. Hodgson et al.

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3. Dose schedule of HU A schedule designed to kill a high proportion of proliferating cells was developed using CFU-S proliferating in the spleens of lethally irradiated mice 1 week after the transplantation of 2 x lo6 cells. One injection of HU (1 mg/g BW) depressed the CFU-S content of the spleen to 50 i-10 %. From the data of the disappearance of HU from blood and its effects on DNA synthesis (Fig. 1), it could be predicted that when a second dose of HU was given up to 3 hr after the first, the HU concentration at the moment of the second injection would be such that no cells would be in S phase, and thus no further killing should occur. A second injection given later than 3 hr after the first would be expected to find a partially synchronized group of cells in S phase, which would be sensitive to the killing effect of the drug. As the interval between doses increased the fraction of cells in S phase should increase, reach a maximum, and then decrease as cells began to leave the S phase. That this occurred is shown in Fig. 2

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FIG.2. Survival of CFU-S proliferating in the spleen of lethally irradiated mice after a second injection of HU. Ordinate A: Surviving fraction. Ordinate B: Fraction of DNA synthesis (‘S) estimated as ‘S’= 1 - surviving fraction. Abscissa: interval betweenHU injections. 0 , Surviving fraction (mean k SE). A, Fraction in DNA synthesis. The two interrupted lines above and below the value of 1 for surviving fraction represents the mean f SE, for CFU-S survival in mice receiving only the first injection of HU.

which summarizes the results of experiments of this kind. Injections of HU up to 3 hr after the first injection produced no further detectable decrease in CFU-S. Then as the interval between doses increased the percentage of CFU-S killed increased, to reach a maximum of 86 between 6 and 8 hr. After an interval of 11 hr, only 50 % of cells were vulnerable to HU, indicating loss of synchrony by progression through the cycle. On the basis of these experiments, two doses of HU, 7 hr apart, were given to normal female mice in an attempt to eliminate most of the proliferating progenitors and leave most of the non-proliferating CFU-S intact.

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Recovery of proliferating haemopoietic progenitor cells

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FIG.3. CFU-S, CFC and total cells in (a) femur, (b) spleen of mice after injection of two doses of 1 mg/g of HU, separated by 7 hr. Ordinate: content as a percentage of non-injected controls. Abscissa: time in days after initiation of HU treatment. 0 , Total cells (mean & SE); 0, total CFU-S content (mean k SE); A , total CFU-C content (mean k SE); x, indicate coincidence between CFU-S and CFU-C.

4. Effects of two doses of HU 7 hr apart on CFC and CFU-S Fig. 3 summarizes the results of the assays of femoral CFU-C in four experiments, and of femoral CFU-S, spleen CFU-S and spleen CFU-C in three experiments. In each experiment, groups of five mice were killed at different intervals after the initiation of HU treatment and each experiment included a group of normal mice. Two hours after the second injection femoral CFU-C (Fig. 3a) averaged 8.5 % while CFU-S were 70 % of controls. Femoral CFU-C rose exponentially thereafter until day 3, with doubling time of 16.5 hr. Femoral CFU-S fell during the first day after initiation of the treatment and reached a value of 40 % at which they remained for another day and then returned to normal during the third day. Marrow cellularity decreased progressively for 2 days after HU, then rose abruptly during day 3. Spleen CFU-C and CFU-S (Fig. 3b) decreased to about 40% 2 hr after the second HU injection, remained at this level for 3 days and then increased to several times normal levels on day 4.These high levels were maintained through day 6 and then declined slowly to return to normal by 13 days (not shown in Fig. 3b). The extent of the CFU-C overshoot (fivefold) was more reproducible (smaller SE) than that of CFU-S, which varied from two- to five-fold between experiments. Spleen cellularity did not decrease as much as that of marrow and like CFU-C and CFU-S recovered to supra normal levels between day 3 and 4 which was 1 day after marrow recovery. 5 . Proliferative state of marrow CFU-S The proliferative state of CFU-S of normal mice as inferred from sensitivity to HU is shown in Table 1. The first injection of H U did not affect CFU-S. A second injection given

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G . S.Hodgson et al. TABLE 1. Effect of hydroxyurea (HU) on marrow CFU-S

Group (Exp., time of the injection before sampling) 1. H U , 2 h r HU, 9,2 hr HU, 9

NBM control 2. HU, 24, 17,2 hr HU, 24,17

NBM 3. HU, 48,41,2 hr HU, 48,41

NBM

SE

CFU/femur

1107 778 1103 1180

93 118 73 101

250 430 999

19 45 165

318 524 1360

26 60 93 ~~

4. HU, 72,65,2 hr HU, 72,65 HU, 96, 89,2 hr HU, 96,89

NBM 5. HU, 144, 137,2 hr HU, 144,137

NBM

717 1620 810 914 1315

78 146 179 101 129

1400 1180 1590

168 118 142

The marrow CFU-S content was assayed in groups of fourteen irradiated mice injected with a known fraction of femoral marrow. In each experiment a group of mice injected with normal bone marrow was included, and another smaller (four or five) group served as irradiation control; of twenty-four irradiated controls, twenty-three had no colonies on the spleen and one had two colonies.

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7 hr after the first significantly decreased CFU-S levels to 70 %. CFU-S remained sensitive to HU for 3 dyas after initiation of treatment and then regained a non-cycling state. 6. Efect of HU on erythropoiesis and on erytliropoietin sensitive cells ESC The effect of HU on erythropoiesis as measured by 59Feincorporation into RBC of female mice is shown in Fig. 4. The 18hr RBC 59Fecontent decreased to below 1 % on day 1, remained low for 2 days and then rose sharply to overshoot by day 4. 48 hr RBC uptake showed a similar response except for the duration of the overshoot which was lower and of shorter duration than that of the 18 hr RBC 59Fe, suggesting a shortened marrow transit time for RBC precursors. Since the RBC precursors which incorporate 59Feare derived from erythropoietin sensitive cells, one would infer on the basis of the data in Fig. 4 and the known time course of response to EPO (Filmanowicz & Gurney, 1961), that the ESC had been eliminated by the two HU injections, that recovery would be under way by day 1, and that there would be an overshoot. Fig. 5 shows the effects of the two HU injections on the response to EPO in groups of seven RBC transfused mice. The results obtained were in agreement with what one would infer

Recovery of proliferating haemopoietic progenitor cells

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FIG.4. Effect of two doses of HU on sgFe incorporation into RBC. 0 , 59Fecontent of RBC measured 18 hr after tracer injection (mean k SE); A , 59Fe content of RBC measured 48 hr after tracer injection (mean k SE). ------, Normal mouse 18 hr 59Fecontent of RBC; ..*-.*., normal mouse 48 hr 59Fecontent of RBC.

from the 59FeRBC curve and showed a nearly complete disappearance of ESC followed by very rapid recovery. The rate of recovery of the responsiveness was such that RBC 59Fe content went from 0.14% k 0.01, when EPO was injected 2 hr after the second HU injection to 9.3 % & 0-6,when it was injected 24 hr after. The recovery during this initial period was studied in a repeat experiment which gave 0.27 % k 0.01 for the initial point and 7.5 % 5 0.8 for the 24 hr point. A doubling time can be calculated for this portion of the recovery curve if it is assumed to be exponential; this turns out to be 5 hr. 7. Effect of HU on circulating blood cells The responses of peripheral blood cell numbers to the two doses of HU are shown in Fig. 6. The packed RBC volume was not significantly affected. The blood reticulocytes declined for 3 days then increased to reach supra normal levels by day 4 as would have been predicted from the 59Fedata. Granulocytes declined in parallel to reticulocytes, but recovery started 1 day later.

DISCUSSION

A two-dose schedule of HU injected into normal mice reduced markedly the levels of proliferating CFU-C and ESC and left the major portion of the CFU-S compartment intact. During the initial phase of CFU-C and ESC recovery, there was a depletion of the CFU-S population, which entered cell cycle and remained in cycle until its numbers were replenished. The rapid entry of CFU-S in bone marrow into S phase after a single injection of HU had previously been noted (Vassort, Frindel & Tubiana, 1971). CFU-S in spleen were depleted in similar

G . S.Hodgson et al.

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FIG.5 . Effects of two doses of HU on the response to 3 u of erythropoietin in RBC transfused mice. Ordinate: response to erythropoietin ("Fe content of RBC measured 24 hr after tracer injection) expressed as a percentage of the response of non HU injected mice (mean k SE). Abscissa: Time of EPO injection after second dose of HU. The iron uptake in transfused non EPO treated mice was 0.42 & 010, and that of EPO treated mice 19.1 f 0.4.

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FIG.6 . Effect of two doses of HU on peripheral blood counts. 0 , RBC packed cell volume mean (SE within the radius of circle); A, reticulocytes (mean 2 SE); 0, granulocyte counts (mean -t SE); ____-_ ,mean normal value for granulocytes and reticulocytes.

Recovery of proliferating haemopoietic progenitor cells

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fashion to marrow but did not recover until after the marrow compartment had returned to normal and then showed a pronounced overshoot. While femoral CFU-C increased exponentially from 2 hr after the second HU injection, spleen CFC, showed no increase till between day 3 and 4 when there was a pronounced rise of both spleen CFU-S and CFU-S to supra normal levels. The rapidity and magnitude of the splenic overshoot of CFU-C and CFU-S on day 4 (an increase of about ten-fold in 1 day) suggests that a major portion of this increase may be due to migration; the mechanism of which is at present unknown. Perhaps infection, as a consequence of the granulocytopenia which is maximum at day 4, may be a contributing factor. Injection of bacterial products is known to increase both spleen CFU-S and CFU-C (McCulloch et al., 1970). The observed findings make it appear as if CFU-S respond quickly to depletion of the proliferating progenitor compartments and feed in cells to these compartments which are thus replenished rapidly. Decrease of CFU-S would appear to trigger the CFU-S compartment promptly into cycle. The time course of events in haemopoietic tissue after the treatment with HU offers an explanation for the observed optimum time spacing of doses of cell cycle dependent cytotoxic drugs as cytosine arabinoside (Skipper, Schabel & Wilcox, 1967)since a 4 day interval would allow recovery of CFU-S numbers and restoration of CFU-S to a non-cycling state, insensitive to cytosine arabinoside. These studies of the effects of HU on cycling CFU-S show how this drug can be used as a synchronizing agent for collecting CFU-S in S phase in vivo in a manner similar to that by Sinclair (1967) for cultured cells in vitro. The change in the fraction of cells in S phase with time after HU injection as found in the two-dose experiment (Fig. 2) with cycling CFU-S, showed that a higher degree of synchronization (about 90 % in S phase) was obtained than that observed by Vassort et al. (1971) for initially non-cycling CFU-S. From the results in Fig. 2 a rough estimate can be made of the duration of the S and G1 + G2 M phases of cyclingCFU-S after one injection of HU. The basic assumption is that in vivo as in vitro (Sinclair, 1967) HU kills cells in S and inhibits DNA synthesis but allows cells in other phases to move through the cycle and accumulate at the G,/S boundary. After HU concentration drops to ineffective levels, between 3 and 4 hr in Fig. 2, the accumulated cells move into S and so become vulnerable again to HU. A mean G2 G1 + M of 4 hr and of S of 7 hr would account for the shape of Fig. 2. Admittedly the value of the duration of S depends heavily on the single 11hr observation, and more experiments are required to evaluate this means of cycle time estimation. The total duration of a cycle is about 11 hr for CFU-S proliferating in the spleen of an irradiated mouse 7 days after injection of marrow cells. This value is like that estimated in a similar fashion by Vassort et al. (1971), following a single dose of HU in mice with non-cycling CFU, but longer than that postulated by Lajtha, Gilbert & Guzman (1971) to occur in the initial phases of CFU-S regeneration. The value proposed (6 hr) is similar to the doubling time found for repopulation of erythropoietic tissue in lethally irradiated mice give lo8 bone marrow cells (Smith, Goodman & Hodgson, 1974) and for regeneration of CFU-E in a lethally irradiated hosts after bone marrow injection (Gregory et al., 1974). Recovery of ESC after H U has a similar doubling time, 5 hr, but it cannot at present be determined how much entry of cells from the nearly normal CFU-S pool and how much limited proliferation of ESC (Reissman & Udupa, 1972) contribute to the rapid recovery.

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G. S.Hodgson et al. ACKNOWLEDGMENTS

The able technical assistance of Miss P. Gilbert, Mrs G. Green and Stuart Haldane is gratefully acknowledged. REFERENCES D. (1966) The growth of mouse bone marrow cells in vitro. Aust. J. exp. Biol. men. BRADLEY, T.R. & METCALF, Sci. 44, 287. BRADLEY, T.R., STANLEY, E.R. & SUMNER, M.A. (1971) Factors from mouse tissue stimulatingcolony growth of mouse marrow cells “in vitro”. Aust. J. exp. Biol. med. Sci. 49, 595. BRADLEY, T.R. & SUMNER, M.A. (1968) Stimulation of mouse bone marrow colony growth “in vitro” by conditioned medium. Aust. J. exp. Biol. med. Sci. 46,607. FILMANOWICZ, E. &GURNEY, C.W. (1961) Studies of erythropoiesis.XVI. Response toasingledoseof erythropoietin in polycythaemicmice. J. Lab. clin. Med. 57, 65. GREGORY, C.J., MCCULLOCH, E.A. &TILL,J.E. (1973) Erythropoieticprogenitors capableof colony formation in culture. State of differentiation.J. CellPhysiol. 41,420. GREGORY, C., TEPPERMAN, A.D., MCCULLOCH, E.A. & TILL,J.E. (1974) Erythropoietin progenitors capable of colony formation in culture. Response of normal and genetically anemic w/wvmice to manipulation of the erythron. J. Cell Physiol. (in press). HODGSON, G. (1967) Effect of vinblastin and 4 amino-N 10 pteroylglutamic acid on the erythropoietin responsive cell. Proc. SOC.exp. Biol. Med. 123,1206. HODCSON, G.S.(1973) Propertiesof haemopoieticstem cells in phenylhydrazinetreated mice. Cell TissueKinet. 6,199. ISCOVE, N.N., TILL,J.E. & MCCULLOCH, E.A. (1970) The proliferative status of mouse granulopoietic progenitor cells. Proc. SOC.exp. Biol. Med. 134, 33. LAJTHA,J., GLLBERT, C.W. & GUZMAN, E. (1971) Kinetics of haemopoietic colony growth. Brit. J. Haemat. 20,343. MCCULLOCH, E.A., THOMPSON, M.W., SIMINOVITCH, L. &TILL,J.E. (1970) Effectsof bacterial endotoxin on haemopoietic colony forming cells in the spleens of normal mice and mice of genotypesI/sld. CellTissue Kinet. 3, 47. MCCULLOCH, E.A. & TILL,J.E. (1972) Leukemia considered as defective differentiation. Complementary in vivo and culture methods applied to the clinical problem. The Nature of Leukemia. Proceedings of the International Cancer Conference, Australian Cancer Society (Ed. by P. C. Vincent), p. 119. MARTIN,R., DONOHUE, D. & FINCH, L. (1972) New analytical procedure for the estimation of DNA with paranitrophenyl hydrazine. Analyt. Biochem. 47,562. MARTIN, R. & HODGSON, G.S. (1973) Estimation of DNA, RNA and lZ5Iand 3H labelled DNA in the same sample. Analyt. Biochem. 52,462. NERY,R. (1966) The colorimetric determination of hydroxamic acids. Analyst, 91, 388. PHILLIPS, E.S., STERNBERG, P., SCHWARTZ, H.S., CRONIN,A.P., SODERGREN, J. & VIDAL, P.M. (1967) Hydroxyurea, acute cell death in proliferating tissues in rats. Cancer Res. 27,61. REISSMAN, K.R. & UDUPA,K.B. (1972) Effect of erythropoietin on proliferation of eythropoietin responsive cells. Cell Tissue Kinet. 5,481. SINCLAIR, W.H. (1967) Hydroxyurea effect on Chinese hamster cells grown in culture. Cancer Res. 27, 297. SKIPPER, H.E., SCHAFIEL, F.M. & WILCOX, W. (1967) Experimental evaluation of anti cancer agents. XXI. Scheduling of arabinosylcytosine to take advantage of its S phase specificity against leukemia cells. Cancer Chemother. Res. 51, 125. Smm, L.H., GOODMAN, G.W. & HODGSON, G.S. (1974) Effectsof isogenicbone marrow cell dose on erythrokinetics of lethally X-irradiated mice. Cell Tissue Kinet. 7,37. STEPHENSON, J.R., AXELRAD, A.A., MCLEOD, L. & SHREEVE, M.M. (1971)Inductionof colonies ofhemoglobin synthesizingcells by erythropoietin in vitro. Proc. nat. Acad. Sci. 68,1542. TILL,J.E. & MCCULLOCH, E.A. (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14,213. VASSORT, F., FRINDEL, E. & TUBIANA, N. (1971) Effectsofhydroxyurea on the kinetics ofcolony forming units of the bone marrow in the mouse. Cell Tissue Kinet. 4,423.

Recovery of proliferating haemopoietic progenitor cells after killing by hydroxyurea.

Cell Tissue Kinet. (1975) 8, 51-60. RECOVERY OF PROLIFERATING HAEMOPOIETIC PROGENITOR CELLS AFTER KILLING BY HYDROXYUREA G . S . HODGSON,T. R. BRADLE...
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